Method of producing an epithelial flap (II)

ABSTRACT

This relates to a lens made of donor corneal tissue suitable for use as a contact lens or an implanted lens, to a method of preparing that lens, and to a technique of placing the lens on the eye. The lens is made of donor corneal tissue that is acellularized by removing native epithelium and keratocytes. These cells optionally are replaced with human epithelium and keratocytes to form a lens that has a structural anatomy similar to human cornea. The ocular lens may be used to correct conditions such as astigmatism, myopia, aphakia, and presbyopia.

RELATED APPLICATIONS

This is a continuation of pending U.S. patent application Ser. No.10/243,121, filed Sep. 13, 2002, that, in turn, is a continuation of PCTApplication No. PCT/US01/22633, having an International Filing Date ofJul. 18, 2001, and in turn, is a continuation-in-part of U.S. patentapplication Ser. No. 09/618,580, filed Jul. 18, 2000, now issued as U.S.Pat. No. 6,544,286, on Apr. 8, 2003.

All of the above disclosures are herein incorporated by reference intheir entirety.

FIELD

This disclosure is in the field of ophthalmology. More particularly, itrelates to a device for lifting an epithelial layer from the anteriorsurface of the cornea. The disclosure includes methods for placement ofa lens beneath that epithelial layer.

BACKGROUND

The visual system allows the eye to focus light rays into meaningfulimages. The most common problem an ophthalmologist or optometrist willencounter is that of spherical ammetropia, or the formation of an imageby the eye which is out of focus with accommodation due to an improperlyshaped globe. The ophthalmologist or optometrist determines therefractive status of the eye and corrects the optical error with contactlenses or glasses.

Many procedures have been developed to correct spherical ammetropia bymodifying the shape of the cornea. Light entering the eye is firstfocused by the cornea, which possesses approximately 75% of the eye'soverall refractory power. The majority of refractive operations involveeither decreasing or increasing the anterior curvature of the cornea.

The procedures in early corneal refractive surgery such as keratophakiaand keratomileusis were originally developed to correct myopia andinvolved removing a corneal disc from the patient with a microkeratome.The removed corneal disc was then frozen prior to reshaping theposterior surface with a cryolathe. After thawing, the disc was returnedto the eye and secured with sutures.

Epikeratophakia, as described in U.S. Pat. No. 4,662,881, is a procedurethat involves inserting a precut donor corneal tissue lens with bevelededges into corresponding grooves in recipient cornea. The lens is thensutured to the corneal bed. The donor lens is lyophilized and requiresrehydration before placement on recipient cornea.

These techniques and their variations were generally considered to beunsuccessful due to frequent complications involving irregularastigmatism, delayed surgical healing, corneal scarring, and instabilityof the refractive result. The problems were attributed to the technicalcomplexity of the procedures as well as to the distortion inarchitecture of the corneal tissue secondary to lens manipulation. Forexample, in epikeratophakia, epithelial irregularity is induced bylyophilization of the donor lens. Freezing of the lenticule inkeratophakia and keratomileusis also causes severe damage to epithelialand stromal cells and disrupts the lamellar architecture of the cornea.

Described is a pre-fabricated lens made of donor corneal tissue obtainedfrom tissue sources such as human or animal cornea. The lens is acorneal disc that is preferably shaped on the posterior surfacegenerally to conform in shape to the eye's anterior surface. The lensmay be shaped by an ablative laser, e.g., by an excimer laser or othersuitable laser. The corneal lenticule is living tissue that has not beenfrozen, lyophilized, or chemically modified, e.g., fixed withglutaraldehyde to crosslink corneal tissue. Pre-existing keratocytes areremoved and then replaced with human keratocytes to decreaseantigenicity. After removal of epithelium in the central zone of therecipient's cornea, the lens is placed on this zone in the same mannerthat a contact lens is placed on the eye.

Ocular lenses found in the prior art do not use native cornea, but areformulated using soluble collagen such as collagen hydrogels, e.g.,polyhydroxyethylmethacrylate, or other biocompatible materials. Forexample, in U.S. Pat. No. 5,213,720, to Civerchia, soluble collagen isgelled and crosslinked to produce an artificial lens. In addition tohydrogels, U.S. Pat. No. 4,715,858, to Lindstrom, discloses lenses madefrom various polymers, silicone, and cellulose acetate butyrate.

In the cases where ocular lenses use corneal tissue, the lenses areeither corneal implants or require a separate agent to adhere the lensto the corneal bed. U.S. Pat. Nos. 5,171,318, to Gibson et al., and5,919,185, to Peyman, relate to a disc of corneal tissue that ispartially or entirely embedded in stroma. The ocular lens devicedisclosed in U.S. Pat. Nos. 4,646,720, to Peyman et al., and 5,192,316,to Ting, is attached to recipient cornea with sutures. The corneal inlaydescribed in U.S. Pat. No. 4,676,790, to Kern, is bonded to recipientcornea using sutures, laser welding, or application of a liquid adhesiveor crosslinking solution.

The ocular lens device described here does not alter the anatomicalstructure of corneal tissue. U.S. Pat. No. 4,346,482, to Tennant et al.,discloses a “living contact lens” consisting of donor cornea that hasbeen anteriorly curved for correction of vision. However, this lens isfrozen prior to reshaping on a lathe which results in stromal keratocytedeath. U.S. Pat. No. 4,793,344, to Cumming et al., also describes adonor corneal tissue lens that is modified by treatment with aglutaraldehyde fixative that preserves the tissue and prevents lensswelling. This treatment alters the basic structure of the corneallenticule by crosslinking the tissue.

Furthermore, the cited documents do not show any methods of lenspreparation that remove native corneal tissue cells and replace themwith cells cultivated from human cornea. My device is devitalized ofnative epithelium and keratocytes to create an acellular corneal tissue,and then revitalized with human epithelium and keratocytes. An attemptto construct a so-called “corneal tissue equivalent” was shown in U.S.Pat. No. 5,374,515, to Parenteau et al. However, the collagen used inthat “equivalent” is obtained from bovine tendon instead of from cornea.The added keratocytes and epithelium are also not from human sources.The tissue using these cell culturing procedures is also quite fragile.

An excimer laser is used to reform a cornea via the “laser in situkeratomileusis” (LASIK) procedure. In this technique, an excimer laseris used to perform stromal photoablation of a corneal flap or in situphotoablation of the exposed stromal bed. Studies have shown that theinaccuracy of correction by this procedure may be as much as one diopterfrom the desired value. Lenses (contacts and spectacles), in contrast,are able to correct within 0.25 diopters of the desired value.

U.S. Pat. No. 6,036,683, to Jean et al., shows the use of a laser toreshape the cornea. However, the laser changes the native structure ofthe cornea by irreversibly coagulating collagen. Post-laser relaxationof collagen is not possible with this treatment.

My described lens, however, in some variations relates to apre-fabricated donor contact lens that adheres to recipient corneawithout sutures. The lens preserves the anatomy of normal cornealtissue. The donor lens may be obtained from human and animal sources, isdevitalized of native keratocytes and epithelium to create an acellulartissue, and then optionally revitalized with at least one of humankeratocytes and epithelial cells to maintain lens viability and decreaseantigenicity. The inventive corneal overlay technique may be completedunder local anesthesia as well as general anesthesia, and theavailability of a precut lens will greatly decrease procedure time,patient cost, and risk of operative complications. The duration ofhealing will also be reduced due to the implementation of a lens alreadyrepopulated with keratocytes.

None of the cited documents shows or suggest the lens and proceduresdescribed herein.

SUMMARY

Described is a pre-fabricated ocular lens device having a lens core madeof donor corneal tissue from tissue sources such as human or animalcornea. The device may be used as a contact lens or as an implanted lensand may have a generally convex anterior surface and, optionally, aconcave posterior surface. The stroma portion of the lens core may berepopulated with replaced keratocytes and the anterior surface ispreferably covered with a replaced epithelium. The lens core adheres torecipient cornea without sutures or other adhering materials.

The lens core may be variously used to correct astigmatism, myopia,aphakia, and presbyopia. The lens core may be made of transgenic orxenogenic corneal tissue. Properly treated, the inventive lens may havea clarity at least 85% of that of human corneal tissue of acorresponding thickness. The lens core is not frozen, lyophilized, orchemically treated with a fixative. However, variations of the devicemay contain therapeutic agents, growth factors, or immunosuppressiveagents.

Another component is a method for preparing the lens device. After sharpdissection of a lenticule from donor corneal tissue, the posteriorsurface is shaped using an ablative laser, such as an excimer laser orother suitable shaping lasers. Native epithelium and keratocytes areremoved and then replaced, as desired, with human epithelium andkeratocytes.

Also described is a method of corneal overlay that involvesde-epithelialization of a portion of the anterior surface of therecipient cornea and placement of the inventive ocular lens device uponthat anterior surface. Another method involves the temporary separationof the epithelial tissue by suction or other procedures and placement ofa lens beneath that epithelial tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a superior, cross-sectional view of the eye.

FIG. 2A is a side view of the focusing point in myopia.

FIG. 2B is a side view of a focusing point corrected by flattening theanterior curvature of the cornea.

FIG. 3A is a side, cross-sectional view of a pre-fabricated donor lens.

FIG. 3B is a side, cross-sectional view of a pre-fabricated donor lenssuitable for correcting myopia.

FIG. 3C is a side, cross-sectional view of a pre-fabricated donor lenssuitable for correcting aphakia.

FIG. 3D is a front view of a pre-fabricated donor lens suitable forbifocal use.

FIG. 3E is a side, cross-sectional view of the FIG. 3C lens positionedaway from the cornea of an eye.

FIG. 3F is a front view of an inventive lens having an overlappingepithelial layer.

FIG. 3G shows a side cross sectional view of the FIG. 3F lens.

FIG. 3H shows a side cross sectional view of an inventive lens in acarrier.

FIG. 3I is a front view of an annular inventive lens.

FIG. 3J shows a side cross sectional view of the FIG. 3I lens.

FIG. 4A is a side, cross-sectional view of an area of de-epithelializedrecipient cornea prepared to receive the optical lens of the presentinvention.

FIG. 4B is a side, cross-sectional view of the donor lens afterplacement on recipient cornea.

FIG. 5 show a series of steps for introducing an inventive lenssubepithelially.

DETAILED DESCRIPTION

The eye is designed to focus light onto specialized receptors in theretina that turn quanta of light energy into nerve action potentials. Asshown in FIG. 1, light rays are first transmitted through the cornea(100) of the eye. The cornea is transparent due to the highly organizedstructure of its collagen fibrils. The margins of the cornea merge witha tough fibrocollagenous sclera (102) and is referred to as thecorneo-scleral layer.

The cornea (100) is the portion of the corneo-scleral layer enclosingthe anterior one-sixth of the eye. The smooth curvature of the cornea isthe major focusing power of images on the retina (104) and it providesmuch of the eye's 60 diopters of converging power. The cornea is anavascular structure and is sustained by diffusion of nutrients andoxygen from the aqueous humor (106). Some oxygen is also derived fromthe external environment. The avascular nature of the cornea decreasesthe immunogenicity of the tissue, increasing the success rate of cornealtransplants.

The cornea consists of five layers. The outer surface is lined bystratified squamous epithelium which is about five cells thick. Failureof epithelialization results in necrosis of the stromal cap andpotential scarring of recipient cornea. The epithelium is supported by aspecialized basement membrane known as Bowman's membrane, which givesthe cornea a smooth optical surface. The bulk of the cornea, thesubstantia propria (stroma), consists of a highly regular form of densecollagenous connective tissue forming thin lamellae. Between thelamellae are spindle-shaped keratocytes which can be stimulated tosynthesize components of the connective tissue. The inner surface of thecornea is lined by a layer of flattened endothelial cells which aresupported by Descemet's membrane, a very thick elastic basementmembrane.

As previously mentioned, the focusing power of the cornea is primarilydependent on the radius of curvature of its external surface. In myopia,as seen in FIG. 2A, increased curvature of the cornea (200) causes thefocusing point of light rays (202) to fall short of the retina (204). InFIG. 2B, flattening the anterior curvature of the cornea (206) correctsthe focal point (208).

Lens Structures

In a first variation of the described lens, the physical shape generallyis of a size and configuration that upon installation on the cornea,supplements the curvature of the cornea to correct abnormal conditionssuch as astigmatism, myopia, hyperopia, presbyopia, and aphakia. Othervariations of the lens may be shaped to be placed beneath the anteriorsurface of the host cornea or to serve as a source of medication.

Typically, the lens core may comprise or consist essentially ofacellular donor corneal tissue that has been devitalized, e.g., treatedto remove native keratocytes and epithelium, to lessen the chances oftissue rejection and then at least partially revitalized, e.g., treatedto introduce at least one of human keratocytes and an epithelial layer,to allow and to support continued use of the inventive lens in place onthe eye. The epithelial cells may (often in the form of a discretelayer) be placed on at least a portion of the anterior surface of alens. In some variations, all of the anterior surface will beso-covered. In one variation discussed below, an epithelial layer willextend beyond the periphery of the lens core and optionally the lens becarried in a biodegradable carrier that is used during placement in theeye and later disappears.

The described lens may be placed on a host eye from which at least amajor portion of the native epithelium on that cornea, has been removed.Preferably in this variation, substantially all of the epithelium hasbeen removed from the region upon which the inventive lens will besited. The lens may also be placed beneath a layer of epithelium liftedfrom the eye surface during the procedure of introducing the lens ontothe anterior surface of the host cornea or in other instances beneaththe surface of the host cornea. The described lens may be used variouslyto correct refraction (because of its shape) or it may be used simply toprovide a source of infused medication to the eye.

The donor lenticule or lens core may be obtained from other human(allogeneic) or foreign tissue (xenogenic) sources. Appropriatexenogenic sources include rabbit, bovine, porcine, or guinea pig cornealtissue. The ocular lens cores may also come from transgenic cornealtissue or corneal tissue grown in vitro. In many instances, it isdesired that the architecture of the corneal layers in the donatedtissue, the normal corneal tissue matrix, e.g., the connective tissue orthe stroma, be substantially preserved. The “corneal tissue matrix” ismade up of thin layers of collagen fibrils. The term “donor cornealtissue”, as used here, is meant to include donor or harvested corneas orcorneal tissue containing the “corneal tissue matrix”. Additionally, inmost variations, it is highly desirable to preserve the anterior surfaceof the donated corneal tissue as found beneath the native epithelium.The donor corneal tissue is not to undergo harsh treatments such aslyophilization, freezing, or other chemical fixation. Nevertheless, itis sometimes desirable to utilize only a portion of the anterior surfaceof the donor lens, e.g., in those instances where the inventive lensstructure is annular in shape.

The described ocular lens device desirably includes Bowman's membrane,where the donor tissue includes it, to maintain the native structure ofhuman epithelium. Again, it is highly desirable to harvest from donorsources in such a way that the native anterior surface below theepithelium is preserved. I have found that these native structures havea superior ability, particularly after the revitalization stepsdiscussed below, to support and to maintain the replaced epithelium alsodiscussed below. The clarity of the inventive tissue lens core handledin such a way generally will be at least 85%, preferably between75%-100%, and most preferably at least 90%, of that of human cornealtissue of corresponding thickness.

The overall diameter of my lens is functionally appropriate to performthe desired correction, and generally is less than about 25 mm and morepreferably is between 10 and 15 mm. The thickness of the resulting lensis, again, functionally appropriate to perform the desired correction,e.g., generally less than 300 μm, more preferably between 5-100 μm.

As shown in FIG. 3B, a lens core (316) for myopic patients is formed,preferably using the procedures discussed below, in such a way that agenerally circular region (318) in the center is flattened in itsanterior curvature. In correction of aphakia, a lens such as is shown inFIG. 3C is formed having a comparatively thicker center (322) and athinner perimeter (324). In general, the shapes discussed here aresimilar to those found in the so-called “soft” contact lenses andinstruction may be had from that technology relating to the overall formof the lenses selected for correcting specific ocular maladies.

As shown in FIGS. 3D and 3E, the lens may also be used to correctpresbyopia. In particular, to treat presbyopia, the lens (330) is alsoprovided with an generally opaque annular region (332) adjacent thecenter of the device. The open center (334) preferably has plano-lenscharacteristics and an effective diameter of less than about 1.5 mm,preferably between about 0.5-1.5 mm, and most preferably between 0.75 mmand 1.75 mm. The diameter of that open center (334) or central area or“pinhole” is generally formed and selected to be less than the pupillarydiameter of the host eye in daylight. This creates a “pinhole” effect,thereby lengthening the overall effective focal length of the eye andminimizing the need for the eye to accommodate. Other bifocal lensdesigns can also be incorporated, e.g., concentric rings, segmented orsectors of the annular region or ring, or progressive diffractive.

FIG. 3E shows a side, cross-sectional view of my lens (330) shown inFIG. 3D, adjacent the anterior surface of a cornea (344) to illustratecertain features of this variation. The outer diameter (336) of theopaque annular ring (332) is generally selected so that it is smallerthan the diameter (338) of the pupil (340) in the iris (342) in lowlight conditions. In this way, the eye's cornea and lens and theinventive lens cooperate in such a way that incident light passes boththough the center of the opaque ring (334), but more importantly, aroundthe periphery of the opaque ring (332), to allow corrected sight duringlow light conditions.

The annular ring (332) may be situated on the lens core either byplacement of a suitable dye, i.e., by “tattooing”, or by placement of asubstantially opaque biocompatible member of, e.g., Dacron mesh or thelike, on the posterior surface to filter light rays. Other placements ofthe annular ring (332) may be envisioned, e.g., on the anterior surfaceof the inventive lens. The annular ring (332) itself preferably is quiteopaque, e.g., passing less than about 80% of incident visible light, butmay be chosen in such a way to be less opaque or to correct othermaladies such as colorblindness by shifting an incident color into avisible range by color refraction or the like.

As is shown in FIGS. 3F (in front view) and 3G (in cross section),another variation of the lens device (346) includes a core lens (348) asdiscussed above but having an epithelial layer (352) that extends beyondthe periphery (350) of that lens core (348). The method for producingthe variation (346) with an extra-periphery epithelial layer (352) issimilar to the method described elsewhere herein except that the lenscore (348) is desirably placed in a carrier (354 in FIG. 3H)) having ashape generally conforming to the anterior surface of the donor corelens (348).

The carrier (354), as shown in FIG. 3H, desirably serves severalfunctions. First, it provides a substrate for growth of the epitheliallayer (352) prior to the time that the core lens (348) is placed on thatepithelial layer (352). This extra surface beyond the periphery of thecore lens (348) provides support for the otherwise fragile epitheliallayer (352). The carrier (354) may be placed in or formed in a properlyshaped receptacle that, in turn, provides support for the fragilecarrier (354) during the steps of growing an epithelial layer (352).

The combination (356) of carrier (354), epithelial layer (352)—whetherthe epithelial layer (352) extends beyond the periphery of the core lens(348) or not, e.g., the epithelial layer (352) is situated only on someor all of the core lens (348)—and core lens (348) placed on thatepithelial layer (352), as shown in FIG. 3H, is another variation of theinvention. The construct (356) shown in FIG. 3H may, upon proper choiceof materials for the carrier, be placed directly in the host eye therebyproviding support for the epithelial layer (352) and core lens (348), aswell as optionally, medication or other treatment materials for the eyeduring initial placement.

When the carrier is used for placement in the eye, the carrier (354)preferably comprises a material meeting two related criteria. First, thematerial desirably is one that dissolves, erodes, or otherwise shortlyclears from the eye to be treated after the combination (356) of thecarrier (354) , epithelial layer (352), and the donor lens (348) areintroduced to that eye. Preferably also, the carrier is of a materialthat serves as a substrate for a pre-grown epithelial layer. Mostdesirably, the carrier (354) satisfies both criteria. The carrier (354)may comprise a material such as collagen, gelatin, starch, glucosamineglucans, proteins, carbohydrates, polyanhydrides such as polylactidesand polyglycolides, their mixtures and copolymers, polydiaxanone, etc.

The carrier (354) may also be infused with medication or other treatmentmaterial, antiangiogenesis materials or the like.

FIGS. 3I and 3J show, respectively, a front view and a side crosssectional view of a lens (360) having a central opening (362) passingthrough the lens body. Although this lens variation (360) is shownwithout an epithelial layer, it is within the scope of the invention toso include the layer.

Process for Shaping the Lens

Returning to FIG. 3A, the donor core lens (300) desirably is obtainedafter slicing corneal tissue from the donor with a microkeratome to formthat lens core (300). The donor lens (300) has a structural surface, theanterior surface of the lens core, which serves as the structuralsurface of the donor corneal tissue. The lens core anterior surface isharvested preferably to retain the Bowman's membrane (where the donorlens contains one) and epithelium (302). The posterior surface (304) ofthe resulting lens is generally concave in shape, although it need notbe so. The anterior surface of the lens may be shaped via a shaping stepwhich preferably involves the use of an ablative laser, such as anexcimer laser, to obtain the necessary power of the lens. Anothersuitable forming step is high pressure water jet cutting.

Sterilization, Devitalization, and Revitalization Steps

Although the order of the process steps outlined below is typical, itshould be understood that such steps may be varied as needed to producethe desired result.

Generally, the lens will first be shaped to an appropriate shape asdiscussed above. The lens core may then be subjected to processes ofsterilization, devitalization, and revitalization. Removal of epithelium(de-epithelialization) and keratocytes (acellularization) from the donorlens will be referred to as “devitalization”. The addition of humanepithelium and keratocytes will be referred to as “revitalization”. Onedesirable method for accomplishing those steps is found just below.Other equivalent methods are known.

Phosphate buffered saline (PBS) with antibiotics, epithelial cell media,and keratocyte media are solutions used during these processes. The “PBSwith antibiotics” solution may contain:

PBS With Antibiotics

1. Amphotericin B (ICN Biomedicals) 0.625 μg/ml

2. Penicillin (Gibco BRL) 100 IU/ml

3. Streptomycin (Gibco BRL) 100 μg/ml

4. Phosphate buffered saline (Gibco BRL)

The composition of the epithelial cell media may include:

Epithelial Cell Media

1. Dulbecco's Modified Eagle Media/Ham's F12 media (Gibco BRL) 3:1

2. 10% fetal calf serum (Gibco BRL)

3. Epidermal growth factor (ICN Biomedicals) 10 ng/ml

4. Hydrocortisone (Sigma-Aldrich) 0.4 μg/ml

5. Cholera toxin (ICN Biomedicals) 10⁻¹⁰ M

6. Adenine (Sigma-Aldrich) 1.8×10⁻⁴ M

7. Insulin (ICN Biomedicals) 5 μg/ml

8. Transferrin (ICN Biomedicals) 5 μg/ml

9. Glutamine (Sigma-Aldrich) 2×10⁻³ M

10. Triiodothyronine (ICN Biomedicals) 2×10⁻⁷ M

11. Amphotericin B (ICN Biomedicals) 0.625 μg/ml

12. Penicillin (Gibco BRL) 100 IU/ml

13. Streptomycin (Gibco BRL) 100 μg/ml

The composition of the keratocyte media may include:

Kerat

1. DMEM

2. 10% neonatal calf serum (Gibco BRL)

3. Glutamine (Sigma-Aldrich) 2×10⁻³ M

4. Amphotericin B (ICN Biomedicals) 0.625 μg/ml

Sterilization Step

After harvesting the lens core from donor corneal tissue and followingthe shaping step, the lens may be sterilized, for instance, by immersioninto 98% glycerol at room temperature. Three weeks of glycerol treatmentinactivates intracellular viruses and any bacteria or fungi. Ethyleneoxide gas sterilization may also be used, but tends to induce variabledamage to stromal tissue.

Devitalization Step

De-epithelialization

I prefer to de-epithelialize the donor lens by placing it in a one molarsolution of salt (preferably sodium chloride) at a temperature from 4 to25° C. After four to eight hours of incubation, the entire epitheliallayer generally will split from the corneal stroma and may be easilyremoved. Thereafter the lens may be washed in a PBS solution withantibiotics to remove salt and cellular material.

Another method of removing the epithelium is via the use of vacuum. Theepithelium may be split from the stroma by means of suction (−100 mm Hgto −450 mm Hg). After fifteen minute to 1 hour, the epithelium typicallywill separate from the stroma at the basement membrane layer. Thereafterthe lens may be washed in a PBS solution with antibiotics to remove saltand cellular material.

Finally, the donor lens may be de-epithelialized by placing it insterile PBS with antibiotics for four hours and changing the solutionmany times. The lens core may then be kept submerged in the PBS solutionat 37° C. for one week to produce a split between the epithelium and thestroma. The epithelium may then be removed, e.g., by physically scrapingor washing with a liquid stream. Small numbers of lenses may be strippedof epithelium by gentle scraping with forceps.

Acellularization

The de-epithelialized lens may be then immersed in a solution ofdetergent (for example 0.025% to 15% sodium dodecyl sulfate) to wash outthe keratocyte cellular material. A detergent will solubilize and washout the keratinocytic material. This can take place from 1 to 8 hours.Afterward the cellular material can be washed in a buffered solutionwith antibiotics to remove detergent and cellular material.

Alternatively, the de-epithelialized lens may be immersed in sterile PBSwith antibiotics for an appropriate period, e.g., several weeks, perhapssix weeks to remove native keratocytes. The solution may be changedtwice weekly. In some instances, it may not be necessary to removekeratocytes from the donor lens, e.g., when the donor tissue is obtainedfrom a transgenic source and has minimal antigenicity.

Revitalization Step

Preparation of Cells

Human epithelial cells and keratocytes are used in the revitalizationprocess. Epithelial cells may be obtained from a tissue bank, but arepreferably obtained from fetal or neonatal tissue. Fetal cells are mostpreferable, since the properties of fetal tissue minimize scarringduring any wound healing process.

In any event, freshly isolated epithelial cells, obtained bytrypsinization of corneal tissue, may be seeded onto a precoated feederlayer of lethally irradiated 3T3 fibroblasts (i.3T3) in epithelial cellmedia. Cells are cultured and media changed every three days until thecells are 80% confluent, about 7-9 days. Residual i.3T3 are removed with0.02% EDTA (Sigma-Aldrich) before the epithelial cells are detachedusing trypsin (ICN Biomedicals). Another method of regeneratingepithelium involves culturing autologous epithelial cells on humanamniotic membrane as described in Tsai et al. (2000). “Reconstruction ofDamaged Corneas by Transplantation of Autologous Limbal EpithelialCells,” New England Journal of Medicine 343:86-93.

Keratocytes may be extracted from the remaining stromal tissue. Thestroma is washed in PBS, finely minced, and placed in 0.5% collagenase A(ICN Biomedicals) at 37° C. for 16 hours. Keratocytes obtained from thisenzyme digest are then serially cultured in keratocyte media. Theepithelial cells and keratocytes generated in the. revitalization stepwill be referred to as “replaced” epithelial cells and keratocytes.

Production of the Donor Lens

The acellular donor lens core may then be placed on a hydrophilic,polyelectrolyte gel for completion of the re-vitalization. The preferredpolyelectrolytes are chondroitin sulfate, hyaluronic acid, andpolyacrylamide. Most preferred is polyacrylic acid. The lens is immersedin keratocyte media and incubated with approximately 3×10⁻⁵ keratocytesfor 48 hours 37° C. Approximately the same amount of epithelial cellsare then added to the anterior stromal surface. Tissue cultureincubation continues for another 48 hours. Keratocyte media is changedevery two to three days. Once the epithelium is regenerated, thepolyelectrolyte gel draws water out of the lens at a pressure of about20-30 mm Hg until the original lens dimensions are obtained.

Replaced epithelium covers at least a portion of the anterior surface ofthis variation of the inventive lens and replaced keratocytes repopulatethe stroma of the lens core after revitalization.

As noted above, another variation of the lens includes an epitheliallayer (352 in FIG. 3G) that extends from the periphery of the lens core(348). The same procedure as just outlined may be used to prepare theepithelial cell layer in the carrier (354) prior to placement of thelens core (348) onto the pre-prepared epithelial cell layer.

It may be beneficial in some instances also to incorporate therapeuticagents, growth factors, or immunosuppressive agents into the lens corefurther to decrease the risk of rejection or remedy disease states.

Placement of the Lens on the Eve

One procedure for applying the lens of this invention is depicted inFIGS. 4A and 4B. During the procedure, the donor lens (300), as shown inFIG. 3A, is placed on a portion of recipient cornea that has beende-epithelialized (308). The result is the placement and construct (312)shown in FIG. 4B. The lens' replaced epithelium and the host epitheliumeventually grow to form a continuous, water-tight layer (310). I havefound that the inventive lens bonds or adheres to the recipient corneawithout sutures or adhesives, but can also be removed withoutsubstantial difficulty.

Another placement procedure variation is shown in FIG. 5. In thisvariation, it is preferable to use a core lens that has been onlypartially revitalized in that the keratocytes have been replaced but theepithelial layer has not. Of course, a core lens that has been partiallycovered with a seed layer of epithelial cells is also acceptable. In anyevent, step a. of FIG. 5 shows a native eye (600) having an epitheliallayer (602) and a corneal stroma (604). Step b. of FIG. 5 shows theplacement of a suction device (606) on the anterior surface of the eye(600). The suction device (606) applies a modest vacuum to theepithelial layer (602), e.g., between about −100 mmHg and −450 mmHg, toraise a section of the epithelial layer (602) as shown in step c. Thisblister (608) typically is filled with a physiologic fluid. Obviously,the suction device (606) has a footprint on the surface of the corneasimilar to the size of the lens to be placed on that cornea. Step d.shows the opened epithelial flap (608) and the placement of the lenstowards the corneal stromal margin (612) beneath that epithelial flap(608). Step c. of FIG. 5 shows the finished placement of the lens (610)on the cornea beneath the native epithelial membrane. This procedure hasa number of benefits including that of being less traumatic to thesurface of the eye than simple removal of the epithelium.

It is also within the scope of the description to use the preparationprocedure for the LASEK procedure for the step of exposing the cornealsurface for application of the lens. The LASEK procedure is known and,unlike the LASIK procedure, does not involve temporary removal of ananterior flap of corneal tissue with a surgical tool but rather onlyutilizes an ethanol wash and a temporary withdrawal of the epitheliallayer for a laser treatment. Such a preliminary step, the washing withethanol to perturb the junction between the corneal stroma and theepithelium is adequate to provide a layer of epithelium for temporarymovement and insertion of the inventive lens on the corneal surface.

I have described the structural and physiologic properties and benefitsof this donor ocular lens. This manner of description should not,however, be taken as limiting the scope of the disclosure in any way.

1. A method for acting on an eye having an anterior corneal surface andan epithelial tissue layer, the method comprising: a.) lifting from theanterior corneal surface, a portion of the epithelial tissue layerresulting in a lifted continuous epithelial layer separated from acorneal stromal margin and having a portion of that lifted continuousepithelial layer connected to the corneal surface, b.) performing anaction on the corneal stromal margin from which a portion of theepithelial layer has been separated, and c.) returning the liftedcontinuous epithelial layer to the eye adjacent to the corneal stromalmargin from which a portion of the epithelial layer has been separated.2. The method of claim 1 wherein the step of performing an action on thecorneal stromal margin comprises placing an ocular device onto thecorneal stromal margin from which a portion of the epithelial layer hasbeen separated.
 3. The method of claim 1 wherein the step of performingan action on the corneal stromal margin comprises placing a correctiveocular device onto the corneal stromal margin from which a portion ofthe epithelial layer has been separated.
 4. The method of claim 1wherein the step of performing an action on the corneal stromal margincomprises performing a laser corrective procedure on the corneal stromalmargin from which a portion of the epithelial layer has been separated.5. The method of claim 1 wherein said lifting step comprises employingvacuum to lift the epithelial layer from the anterior surface.
 6. Amethod for acting on an eye having an anterior corneal surface and anepithelial tissue layer, the method comprising: a.) lifting from theanterior corneal surface with a device, a portion of the epitheliallayer resulting in a living, lifted continuous epithelial layerseparated from a corneal stromal margin having a portion connected tothe corneal surface, and b.) removing the device and leaving theseparated epithelial layer connected to the corneal surface.
 7. Themethod of claim 6 further comprising the step of performing an action onthe corneal stromal margin from which a portion of the epithelial layerhas been separated.
 8. The method of claim 6 further comprising the stepof returning the lifted continuous epithelial layer to the eye adjacentto the corneal stromal margin from which a portion of the epitheliallayer has been separated.
 9. The method of claim 7 wherein the step ofperforming an action on the corneal stromal margin comprises placing anocular device onto the corneal stromal margin from which a portion ofthe epithelial layer has been separated.
 10. The method of claim 7wherein the step of performing an action on the corneal stromal margincomprises placing a corrective ocular device onto the corneal stromalmargin from which a portion of the epithelial layer has been separated.11. The method of claim 7 wherein the step of performing an action onthe corneal stromal margin comprises performing a laser correctiveprocedure on the corneal stromal margin from which a portion of theepithelial layer has been separated.
 12. The method of claim 7 whereinsaid lifting step comprises employing vacuum to lift the continuousepithelial layer separated from the corneal stromal margin.
 13. Themethod of claim 1 wherein the continuous epithelial layer separated froma corneal stromal margin contains substantially no corneal tissue.
 14. Amethod for acting on an eye having a cornea and epithelial tissueattached thereto, the method comprising: a) separating from the cornea atissue layer comprising epithelial tissue substantially free of othercorneal tissue resulting in an exposed anterior corneal surface and aseparated continuous epithelial tissue layer separated from the exposedanterior corneal surface, the separated layer having a portion connectedto the eye, b) placing a corrective ocular device entirely onto theexposed anterior corneal surface, and c) placing at least a portion ofthe separated layer onto the corrective ocular device.
 15. The method ofclaim 14 wherein the separating step comprises employing vacuum to liftthe tissue layer comprising epithelial tissue from the cornea.
 16. Themethod of claim 14 wherein the corrective ocular device further containsat least one of a therapeutic agent, an immunosuppressive agent, and oneor more growth factors.
 17. The method of claim 14 wherein thecorrective ocular device comprises a lens.
 18. The method of claim 14wherein the separating step separates from the cornea a tissue layerconsisting essentially of epithelial tissue.
 19. The method of claim 14wherein the separating step separates from the cornea a tissue layer ofsubstantially only epithelial tissue.
 20. The method of claim 14 whereinthe separating step comprises separating the tissue layer from Bowman'sMembrane.
 21. A method for acting on an eye having a cornea andepithelial tissue attached thereto, the method comprising: a) separatingfrom the cornea with a device, a tissue layer comprising epithelialtissue substantially free of other corneal tissue to produce an exposedanterior corneal surface, and a lifted, living, continuous epithelialtissue layer separated from the exposed anterior corneal surface andhaving a portion connected to the eye, b) removing the device from theeye leaving the lifted epithelial tissue layer having a portionconnected to the eye, c.) introducing a corrective ocular deviceentirely onto the exposed anterior corneal surface, and c) placing atleast a portion of the lifted layer onto the corrective ocular device.22. The method of claim 21 wherein the using step comprises employing avacuum to lift the tissue layer comprising epithelial tissue from thecornea.
 23. The method of claim 21 wherein the corrective ocular devicefurther contains at least one of a therapeutic agent, animmunosuppressive agent, and one or more growth factors.
 24. The methodof claim 21 wherein the corrective ocular device comprises a lens. 25.The method of claim 21 wherein the separating step separates from thecornea a tissue layer consisting essentially of epithelial tissue. 26.The method of claim 21 wherein the separating step separates from thecornea a tissue layer of substantially only epithelial tissue.
 27. Themethod of claim 21 wherein the separating step comprises separating thetissue layer from Bowman's Membrane.
 28. A method for acting on an eyehaving a cornea and epithelial tissue attached thereto, wherein a tissuelayer comprising epithelial tissue substantially free of other cornealtissue has been separated from the cornea, resulting in an exposedanterior corneal surface and a separated continuous epithelial tissuelayer separated from the cornea and the separated layer having a portionconnected to the eye, the method comprising: a) placing a correctiveocular device entirely onto the exposed anterior corneal surface, and b)placing at least a portion of the separated layer onto the correctiveocular device.
 29. The method of claim 28 wherein the corrective oculardevice further contains at least one of a therapeutic agent, animmunosuppressive agent, and one or more growth factors.
 30. The methodof claim 28 wherein the corrective ocular device is a lens.
 31. Themethod of claim 28 wherein the separated layer consists essentially ofepithelial tissue.
 32. The method of claim 28 wherein the separatedlayer is substantially only epithelial tissue.
 33. The method of claim28 wherein the separated layer is free of Bowman's Membrane.
 34. Amethod for acting on an eye having a cornea and epithelial tissueattached thereto, wherein a tissue layer comprising epithelial tissuesubstantially free of other corneal tissue has been separated from thecornea, resulting in an exposed anterior corneal surface and a separatedcontinuous epithelial tissue layer separated from the cornea and theseparated layer having a portion connected to the eye, and a correctiveocular device has been placed entirely onto the exposed anterior cornealsurface, the method comprising the step of: placing at least a portionof the separated layer onto the corrective ocular device.
 35. The methodof claim 34 wherein the corrective ocular device further contains atleast one of a therapeutic agent, an immunosuppressive agent, and one ormore growth factors.
 36. The method of claim 34 wherein the correctiveocular device comprises a lens.
 37. The method of claim 34 wherein theseparated layer consists essentially of epithelial tissue.
 38. Themethod of claim 34 wherein the separated layer is substantially onlyepithelial tissue.
 39. The method of claim 34 wherein the separatedlayer is free of Bowman's Membrane.
 40. A method for acting on an eyehaving a cornea and epithelial tissue attached thereto, the methodcomprising the step of separating from the cornea a tissue layercomprising epithelial tissue substantially free of other corneal tissue,the separating step producing an exposed anterior corneal surface and aseparated continuous epithelial tissue layer separated from the exposedanterior corneal surface, the separated layer having a portion connectedto the eye.
 41. The method of claim 40 further comprising the step ofperforming an action on the corneal stromal margin from which theseparated continuous epithelial tissue layer has been separated.
 42. Themethod of claim 41 wherein the step of performing an action on thecorneal stromal margin comprises placing an ocular device onto thecorneal stromal margin from which the separated continuous epithelialtissue layer has been separated.
 43. The method of claim 41 wherein thestep of performing an action on the corneal stromal margin comprisesplacing a corrective ocular device onto the corneal stromal margin fromwhich the separated continuous epithelial tissue layer has beenseparated.
 44. The method of claim 41 wherein the step of performing anaction on the corneal stromal margin comprises performing a lasercorrective procedure on the corneal stromal margin from which theseparated continuous epithelial tissue layer has been separated.
 45. Themethod of claim 41 further comprising the step of returning theseparated continuous epithelial tissue layer to the eye adjacent to thecorneal stromal margin from which a portion of the epithelial layer hasbeen separated.
 46. The method of claim 42 further comprising the stepof returning the separated continuous epithelial tissue layer to the eyeadjacent to the corneal stromal margin from which a portion of theepithelial layer has been separated.
 47. The method of claim 43 furthercomprising the step of returning the separated continuous epithelialtissue layer to the eye adjacent to the corneal stromal margin fromwhich a portion of the epithelial layer has been separated.
 48. Themethod of claim 44 further comprising the step of returning theseparated continuous epithelial tissue layer to the eye adjacent to thecorneal stromal margin from which a portion of the epithelial layer hasbeen separated.
 49. The method of claim 40 wherein the separating stepcomprises employing vacuum to lift the tissue layer comprisingepithelial tissue from the cornea.
 50. The method of claim 43 whereinthe corrective ocular device further contains at least one of atherapeutic agent, an immunosuppressive agent, and one or more growthfactors.
 51. The method of claim 43 wherein the corrective ocular devicecomprises a lens.
 52. The method of claim 40 wherein the separating stepseparates from the cornea a tissue layer consisting essentially ofepithelial tissue.
 53. The method of claim 40 wherein the separatingstep separates from the cornea a tissue layer of substantially onlyepithelial tissue.
 54. The method of claim 41 wherein the separatingstep comprises separating the tissue layer from Bowman's Membrane.
 55. Amethod for acting on an eye having a cornea and epithelial tissueattached thereto, wherein a tissue layer comprising epithelial tissuesubstantially free of other corneal tissue has been separated from thecornea, resulting in an exposed anterior corneal surface and a separatedcontinuous epithelial tissue layer separated from the cornea and theseparated layer having a portion connected to the eye, the methodcomprising the step of performing a vision corrective action upon theexposed anterior corneal surface.
 56. The method of claim 55 wherein thestep of performing an action on the corneal stromal margin comprisesplacing an ocular device onto the corneal stromal margin from which theseparated continuous epithelial tissue layer has been separated.
 57. Themethod of claim 55 wherein the step of performing an action on thecorneal stromal margin comprises placing a corrective ocular device ontothe corneal stromal margin from which the separated continuousepithelial tissue layer has been separated.
 58. The method of claim 55wherein the step of performing an action on the corneal stromal margincomprises performing a laser corrective procedure on the corneal stromalmargin from which the separated continuous epithelial tissue layer hasbeen separated.
 59. The method of claim 55 further comprising the stepof returning the separated continuous epithelial tissue layer to the eyeadjacent to the corneal stromal margin from which a portion of theepithelial layer has been separated.
 60. The method of claim 56 furthercomprising the step of returning the separated continuous epithelialtissue layer to the eye adjacent to the corneal stromal margin fromwhich a portion of the epithelial layer has been separated.
 61. Themethod of claim 57 further comprising the step of returning theseparated continuous epithelial tissue layer to the eye adjacent to thecorneal stromal margin from which a portion of the epithelial layer hasbeen separated.
 62. The method of claim 58 further comprising the stepof returning the separated continuous epithelial tissue layer to the eyeadjacent to the corneal stromal margin from which a portion of theepithelial layer has been separated.
 63. The method of claim 57 whereinthe corrective ocular device further contains at least one of atherapeutic agent, an immunosuppressive agent, and one or more growthfactors.
 64. The method of claim 57 wherein the corrective ocular devicecomprises a lens.
 65. The method of claim 55 wherein the separatedcontinuous epithelial tissue layer consists essentially of epithelialtissue.
 66. The method of claim 55 wherein the separated continuousepithelial tissue layer is substantially only epithelial tissue.
 67. Themethod of claim 55 wherein the separated continuous epithelial tissuelayer is free of Bowman's Membrane.